(1) Field of the Invention
The present invention relates to a thin-layer chromatography flame ionization detector for carrying out a quantitative analysis of chromatographically-separated substances obtained from a sample solution in a thin-layer chromatography system.
(2) Description of the Related Art
In the field of chromatography, the use of a flame ionization detector (FID) for carrying out a quantitative analysis of chromatographically-separated substances obtained from a sample of a mixture in chromatography systems is well known.
For example, a flame ionization detector used in the field of gas chromatography comprises a gas burner connected to a hydrogen gas source and to a chromatographic column through which a sample of mixed gas is passed, a collector electrode disposed above a hydrogen flame formed by the nozzle of the hydrogen gas burner, an electric source for applying a voltage between the hydrogen gas burner and the collector electrode, an amplifier for detecting and amplifying an electric current generated at the collector electrode, and a recorder connected to the amplifier for recording an output obtained therefrom as a chromatogram.
In operation, the sample gas is passed through the chromatographic column in such a manner that it is chromatographically separated into at least two kinds of gaseous substances, of which the sample gas is composed. The separated substances, which are successively fed out of the chromatographic column, are fed into the hydrogen ga burner and are mixed with hydrogen gas fed therein from the hydrogen gas source. Thus, the separated substances are successively ejected together with hydrogen gas from a nozzle port of the hydrogen gas burner, and are burned and ionized by the hydrogen flame formed by the nozzle port of the hydrogen gas burner. A voltage is applied between the collector electrode and the hydrogen gas burner by the electric source and the collector electrode is exposed to the burned and ionized gas so that an ionization current is generated at the collector electrode. The ionization current is detected and amplified by the amplifier and is then recorded as a chromatogram by the recorder.
The value of the ionization current generated in the collector electrode on each of the separated substances depends upon the amount of ionized gas obtained by the burning of the corresponding one of the separated substances. In other words, the amount of each of the separated substances corresponds to an output value obtained from the amplifier at the corresponding one of the separated substances. This makes it possible for the separated substances to be quantitatively analyzed by using a calibration characteristic representing a relationship between a known amount of each of the separated substances and an output value obtained from the amplifier at the known thereof.
In the flame ionization detector used in the field of gas chromatography, since the calibration characteristic representing a relationship between an amount of each of the separated substances and an output value obtained from the amplifier thereat can be obtained as a linear function, it is possible to carry out an accurate quantitative analysis with a good reproducibility.
In the field of thin-layer chromatography, also it is known that a flame ionization detector can be used for a quantitative analysis of chromatographically-separated substances obtained from a sample solution in a thin-layer chromatography system. This thin-layer chromatography flame ionization detector is similar in essence to that used in the field of gas chromatography.
In the thin-layer chromatography system concerned, a thin-layer chromatographic element is used, as disclosed in Examined Japanese Patent Publication No. 52-35320 (Patent No. 907248), which forms a part of a thin-layer chromatograph. The thin-layer chromatographic element comprises a rod having a diameter of about 0.8 to 1.0 mm and made of a refractory material such as silica glass, and a thin-layer formed on the rod by coating the surface thereof with an inorganic absorbent material such as silica gel, alumina, diatomite or the like. A sample solution is spotted on the rod-like element or the thin-layer chromatographic element and is then developed along a length of the rod-like element with a developing solvent, in the same manner as used in a known thin-layer chromatography system, whereby the sample solution is chromatographically separated into at least two kinds of substances, which appear to form at least two zone sections on the rod-like element.
In the operation of the thin-layer chromatography flame ionization detector, the rod-like element carrying the developed and separated substances is gradually passed through the hydrogen flame formed by the hydrogen gas burner, so that these substances are burned and ionized thereby. Thus, the collector electrode is exposed to the burned and ionized gas so that an ionization current is generated at the collector electrode. The ionization current is detected and amplified by the amplifier and is then recorded as a chromatogram by the recorder. Namely, in the flame ionization detector used in the field of thin-layer chromatography, a quantitative analysis of each of the separated substances can be also carried out in substantially the same manner as that used in the field of gas chromatography.
In the thin-layer chromatography flame ionization detector, it is also necessary to prepare a calibration characteristic representing a relationship between an amount of each of the separated substances and an output value obtained from the amplifier on the amount thereof, before carrying out a quantitative analysis of the separated substances, but it is impossible to obtain such a calibration characteristic as a linear function In other words, the calibration characteristic is similar to an exponential function, and accordingly, the thin-layer chromatography flame ionization detector possesses various drawbacks, and little attempt has been made to overcome these drawbacks.
This is because, first, it is very difficult to obtain calibration characteristics on substances to be chromatographically separated, since many plots must be prepared to obtain each calibration characteristic due to the curving thereof. In other words, in order to obtain the calibration characteristics, it is necessary to seek output values of the amplifier which correspond to many kinds of known amounts of each of the substances, because the calibration characteristic so obtained is not linear but curved.
As another drawback, it is impossible to expect a uniform precision in a quantitative analysis of chromatographically-separated substances because of an uneven, therefore, nonuniform gradient of the calibration characteristic curves.
Furthermore, it is very difficult to carry out a reliable quantitative analysis of chromatographically-separated substances because of the distinctiveness of the calibration characteristic curves. In particular, a quantitative analysis of chromatographically-separated substances is frequently carried out in such a manner that each of the separated substances is quantitatively evaluated from the calibration characteristic curve thereof not as an absolute amount but as a relative amount, as in other quantitative analysis fields. In this case, the ratios among the output values of the amplifier which correspond to the separated substances, respectively, are quantitatively evaluated as a relative amount, but these ratios are affected by the amount of a sample solution spotted on the thin-layer chromatographic element, because of the distinctiveness of the calibration characteristic curves. In other words, it is necessary to spot the same amount of a sample solution at all times on the rod-like element, to obtain a reliable quantitative evaluation of the separated substances. However, since it is very difficult or substantially impossible to spot exactly the same amount of a sample solution at all times on the rod-like element, the reproducibility of the quantitative analysis, wherein the chromatographically-separated substances are quantitatively evaluated as a relative amount, are poor.
Namely, in the prior flame ionization detector used in the field of thin-layer chromatography, an accurate quantitative analysis cannot be carried out with a good reproducibility because of the non-linearity of the calibration characteristics.
According to the research by the inventor, it can be assumed that the non-linearity of the calibration characteristics occurs for the following reasons:
When the rod-like element carrying the chromatographically-separated substances is passed through the hydrogen flame, the element is charged with electricity in accordance with an electric potential distribution between the collector electrode and the nozzle of the hydrogen gas burner, so that the rod-like element has a negative polarity to that of the collector electrode. As a result, positive ions caused by the burning and ionization of the separated substances collide with the rod-like element, thereby causing a propagation of electrons and distorting the linearity of the calibration characteristics. The grounds for this assumption will be explained in detail hereinafter.